• Journal of Pharmaceutical Investigation
• /
• v.40 no.3
• /
• pp.175-180
• /
• 2010

Albumin-modification has been viewed as one of the most effective ways of extending the short in vivo lifetimes of peptide drugs by delaying glomerular filtration. In this study, we describe a new type 2 anti-diabetic exendin-4 (Ex4) peptide derivative with significant binding ability to human serum albumin (HSA). This exendin-4 derivative consists of a 4-branched polyethylene glycol $(PEG)_{5k}$ (Mw: 20 kDa) modified with three stearylamines ($C_{18}-NH_2$) and one exendin-4 on its branches. PEG and stearylamine were selected to provide functionality to increase molecular size and bind to albumin, respectively. This derivative ($3C_{18}-4PEG_{5k}$-Ex4) was shown to have larger molecular size (Ca. 152 kDa) than actual (25.0 kDa) when subjected to size-exclusion chromatography, and the fluorescein-tagged $3C_{18}-4PEG_{5k}$-Ex4 displayed significant binding to the HSA-immobilized Sepharose CL-4B resin using confocal laser scanning microscopy. Furthermore, $3C_{18}-4PEG_{5k}$-Ex4 was found to have acceptable anti-hyperglycemic efficacy via three consecutive oral glucose tolerance testings (OGTT) in fasted type 2 diabetic db/db mice. The $HD_{total}$ value ($57.6{\pm}12.3%$) of $3C_{18}-4PEG_{5k}$-Ex4 at a 50 nmol/kg dose was 2-fold greater than that ($31.0{\pm}8.7%$) of native exendin-4 in non-fasted db/db mice. Especially, the blood glucose levels in the mice group treated with $3C_{18}-4PEG_{5k}$-Ex4 did not rebound to ~150 mg/dL until 24 h after the injection, which obviously shows the extended hypoglycemia. We believe that this derivative has great pharmaceutical potential as a novel long-acting type 2 anti-diabetic injection treatment.

• Macromolecular Research
• /
• v.8 no.1
• /
• pp.44-52
• /
• 2000

Styrenic macromonomers with/without a silyloxy-functional group were synthesizedvia chain-end functionalization using 4-vinylbenzyl chloride as a terminating agent insec-butyllithium-initiated polymerization of styrene. The yields were 92 mol% for the silyloxy group and 88 mol% for the styrenic unit. Crystalline polystyrene-g-amorphous polystyrenes were synthesized by (η$^{5}$ -indenyl)-trichlorotitanium ((Ind)TiCl$_3$)-catalyzed copolymerizations of the macromonomers with styrene in the presence of methyl-aluminoxane (MAO) in toluene at 4$0^{\circ}C$. The macromonomer having $\alpha$, $\alpha$'-bis (4-[tert-butyldimethylsilyl-oxy]phenyl) group was also utilized for the preparation of a precursor of hydroxyl-functionalized syndio-tactic polystyrene. The obtained polymers were characterized by a combination of$^1$H, $^{13}$ C NMR spectroscopic, size exclusion chromatographic, and differential scanning calorimetric analysis. The (Ind)TiCl$_3$-catalyzed copolymerization of styrene with the macromonomer carrying the silyloxy functional group was found to be an efficient method to modify syndiotactic polystyrene without a great loss of physica] property by controlling the feud ratio of the macromonomer.

• Journal of the Korean Chemical Society
• /
• v.42 no.2
• /
• pp.190-196
• /
• 1998

A new sampling and size exclusion chromatography (SEC) method for the analysis of underivatized cellulose are established. In this method, cellulose materials are first dissolved in N-methylmorpholine N-oxide (NMMO) and diluted by adding dimethyl sulfoxide (DMSO) to make the sample solutions of about 0.1% in 50/50 NMMO/DMSO (w/w). Sample solutions are analyzed using a glucose-treated divinylbenzene (DVB) SEC column and DMSO containing 0.05M LiBr and 2.5 blank as the eluant. The flow rate was constant at 1 mL/min and the whole SEC system including the column was heated at $80^{\circ}C$ to reduce the viscosity of DMSO. Addition of 0.05 M LiBr eliminated SEC baseline drifting, and addition of 2.5 blank seems to reduce the interaction between the sample and the column packing. SEC molecular weights were determined using a calibration curve constructed from a series of narrow pullulan standards, and they were used to measure the degree of degradation during two different pulp-to-sponge processings.

• BMB Reports
• /
• v.39 no.3
• /
• pp.270-277
• /
• 2006

Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba $\delta$-endotoxin have been shown to be important determinants for mosquito-larvicidal activity, likely being involved in membrane-pore formation. In this study, the Cry4Ba mutant protein containing an additional engineered tryptic cleavage site was used to produce the $\alpha4$-$\alpha5$ hairpin peptide by an efficient alternative strategy. Upon solubilization of toxin inclusions expressed in Escherichia coli and subsequent digestion with trypsin, the 130-kDa mutant protoxin was processed to protease-resistant fragments of ca. 47, 10 and 7 kDa. The 7-kDa fragment was identified as the $\alpha4$-loop-$\alpha5$ hairpin via N-terminal sequencing and mass spectrometry, and was successfully purified by size-exclusion FPLC and reversed-phase HPLC. Using circular dichroism spectroscopy, the 7-kDa peptide was found to exist predominantly as an $\alpha$-helical structure. Membrane perturbation studies by using fluorimetric calcein-release assays revealed that the 7-kDa helical hairpin is highly active against unilamellar liposomes compared with the 65-kDa activated full-length toxin. These results directly support the role of the $\alpha4$-loop-$\alpha5$ hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.

• Tribology and Lubricants
• /
• v.31 no.5
• /
• pp.195-204
• /
• 2015

Several researches are focused on improving the value of fine chemicals based on biomass resources due to environmental and other concerns associated with the use of petroleum-based products. Therefore, the synthesis and application of estolides derived from plant-based waste oil materials and their application as lubricants and as processing oil for butyl rubber products have been studied. Four kinds of estolide were prepared with conversions of 71~92% over 24h using various vegetable oils, as determined by size exclusion chromatography (SEC) and nuclear magnetic resonance (NMR) spectroscopy. FT-IR spectroscopy determines the esterification of estolides using 2-ethylhexyl alcohol. The estolides have iodine values of 35~90, α-ester/α-acid ratios of 0.45~0.55, and total acid number of 114~134 mg KOH g^–1. Four ball wear tests show that the wear scar diameters (WSDs) of estolides as base oil significantly decreased to 0.328~0.494 mm, compared to WSDs of 0.735 and 0.810 mm of WSD for 150N and Yubase 6, respectively, as general base oil. Thus, the estolides have better wear resistance and satisfying design objectives for the engineering of a variety of lubricant base oils.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.41 no.4
• /
• pp.456-464
• /
• 1996

Fructans are the major vegetative storage carbohydrate in wheat(Triticum aestivum L.). The depolymerization of fructans occurs by the sequential removal of terminal fructosyl residues by a specific fructan exohydrolase(FEH). The objective of this study was to isolate and characterize this enzyme in wheat. From stems and sheaths of field-grown wheat(cv. Clark), FEH was purified 356-fold using salt precipitation and a series of chromatographic procedures including size exclusion, anion exchange, and affinity chromatography. FEH had a molecular weight of 63.7 kD and an optima at pH 5.5 and 3$0^{\circ}C$. The $K_{m}$s for $\beta$(2 longrightarrow1) linked oligofructans varied, from 10 to 37mM, with the lowest $K_{m}$ for tetrasaccharide. The $V_{max}$ increased as degree of polymerization (DP) increased. Wheat FEH hydrolyzed only, $\beta$(2 longrightarrow1) linked fructans but not, $\beta$(2 longrightarrow6) linked timothy fructan or sucrose. The role of this FEH in fructan metabolism in wheat is discussed.sed.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.6
• /
• pp.1250-1257
• /
• 2005

Two groups of exopolysaccharides (designated as Fr-I EPS and Fr-II EPS) were isolated from the culture filtrate of new fungal strain Trichoderma erinaceum DG-312 by Sepharose CL-6B chromatography. The structures of the exopolysaccharides were investigated using gas chromatography (GC), Fourier transform-infrared (FT-IR) spectroscopy, GCMS analysis, and NMR. GC analysis indicated that Fr-I EPS was composed of mainly mannose ($78.9\%$) and galactose ($21.1\%$), whereas Fr-II EPS contained mannose ($68.4\%$), galactose ($26.2\%$), and glucose ($5.4\%$). In the anomeric region ($950-700cm_{-1}$) of the FT-IR spectrum, both EPSs exhibited obvious characteristic absorption of $810\;cm_{-1}$, indicating the existence of mannose. The spectra of $\alpha-and\;\beta$-configurations were assigned at 880 and $914\;cm_{-1}$, respectively. The results of GC-MS analyses confirmed that both EPSs were complex heteropolysaccharides with a ($1{\rightarrow}3$)-linked mannan backbone. The C-1 region that appeared in the $^{13}C-NMR$ spectra of these EPSs indicated a typical anomeric carbon signal. The Fr-I EPS showed two anomeric carbon signals at 102.6 and 99.6 ppm, whereas the Fr-II EPS displayed four anomeric carbon signals at 102.5, 99.6, 98.5, and 94.3 ppm. The molecular characteristics of the EPSs were further investigated using a size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS) system. The SEC/MALLS system revealed that the average molar masses of the EPSs were $6.592{\times}10^{4}$ (Fr-I EPS) and $1.920{\times}10^{4}$ (Fr-II EPS) g/mol, and the molecular conformation of both EPSs in aqueous solution was random coils.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.10
• /
• pp.1201-1205
• /
• 2009

Corn starches with different amylose contents were enzymatically modified using Thermus aquaticus 4-$\alpha$-glucanotransferase ($TA{\alpha}GTase$). Upon the enzyme treatment, the chain-length distributions of isoamylolytically debranched products became broader [degree of polymerization (DP): 3-40] than those of untreated corn starches. In addition, a variety of cycloamyloses (CAs) with different sizes were formed by the glucanotransfer activity of $TA{\alpha}GTase$. CAs with DP 5-40 were detectable in all of the $TA{\alpha}GTase$-treated corn starches. From the results of high-performance anion-exchange chromatography and high-performance size-exclusion chromatography analyses, it was suggested that the amount of CAs produced by the enzyme treatment increased as the amylose content of the starches increased. Thus, we concluded that the extent of modification of starch molecules was enhanced in proportion to amylose content by the transfer activity of $TA{\alpha}GTase$. This finding could be useful for developing an efficient process of CA production using this enzyme.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.2
• /
• pp.271-278
• /
• 2020

Glycerol dehydrogenase (GlyDH) catalyzes the oxidation of glycerol to dihydroxyacetone (DHA), which is the first step in the glycerol metabolism pathway. GlyDH has attracted great interest for its potential industrial applications, since DHA is a precursor for the synthesis of many commercially valuable chemicals and various drugs. In this study, GlyDH from Klebsiella pneumoniae (KpGlyDH) was overexpressed in E. coli and purified to homogeneity for biochemical and molecular characterization. KpGlyDH exhibits an exclusive preference for NAD⁺ over NADP⁺. The enzymatic activity of KpGlyDH is maximal at pH 8.6 and pH 10.0. Of the three common polyol substrates, KpGlyDH showed the highest k_cat/K_m value for glycerol, which is three times higher than for racemic 2,3-butanediol and 32 times higher than for ethylene glycol. The k_cat value for glycerol oxidation is notably high at 87.1 ± 11.3 sec^-1. KpGlyDH was shown to exist in an equilibrium between two different oligomeric states, octamer and hexadecamer, by size-exclusion chromatography analysis. KpGlyDH is structurally thermostable, with a T_m of 83.4℃, in thermal denaturation experiment using circular dichroism spectroscopy. The biochemical and biophysical characteristics of KpGlyDH revealed in this study should provide the basis for future research on its glycerol metabolism and possible use in industrial applications.

• THE INTERNATIONAL COMMERCE & LAW REVIEW
• /
• v.19
• /
• pp.7-31
• /
• 2003

The governments of almost countries have the rules and procedures that the purchasing entities have to follow, in order to ensure that the best value for money spent is obtained in procuring goods and services. However, there are often some of important problems in their rules relating to government procurement. Namely, almost countries have a variety of discriminatory regulations for foreign suppliers in the government procurement laws with the object of national security, economic welfare, and protection of domestic market from international competition. For this reason, several advanced countries had reached the Plurilateral Agreement on Government Procurement during Tokyo Round(1973-1979) and Uruguay Round(1986-1994). However, the provisions of two agreements do not apply to all products made by the government but only to those made by purchasing entities specified by each member country in its list in the Appendix. Accordingly, the size of goods and services purchased from foreign suppliers were comparatively not large. As we know well, the United States have spent a large amount of money from federal and state government budget. But the portion of procurement from foreign suppliers is still small, compared with the portion of procurement from domestic suppliers. The main reason are that U.S. has applied for long time the so-called Buy American Act to government procurement positively and maintained many kinds of other domestic regulations which have discriminatory provisions for foreign goods and foreign suppliers. On the recognition of these points, this thesis deals with the Buy American Act and other U.S. domestic laws, regional and bilateral, and plurilateral agreements including WTO Agreement on Government Procurement. As a result, the author found that there are several concerns and problems in the U.S. regulations relating to government procurement. It include the provisions on priority procurement of U.S.-produced products, local contents requirements, set-aside procurement from small business, and exclusion of preferential procurement from the developing countries.